Oleaginous yeast such as Yarrowia lipolytica have the natural ability to use glucose as their sole carbon source; however, this substrate is not always the most cost-effective carbon source. Using sucrose as a carbon source (whether alone or in combination with other carbon sources) as a carbon source instead of glucose may be advantageous due to its cost.
Y. lipolytica is not able to utilize sucrose as a carbon source since it does not have a gene encoding invertase, which catalyzes the conversion of sucrose (a disaccharide) into the monosaccharides glucose and fructose. Several previous investigators have fused a signal sequence to a heterologous gene encoding invertase (e.g., the Saccharomyces cerevisiae SUC2 gene), to engineer the yeast to secrete a mature invertase protein into the surrounding medium, where sucrose can then be hydrolyzed.
One well known signal sequence isolated from Y. lipolytica is that of the inducible alkaline extracellular protease (“AEP”) (EP0220864 B1; Davidow, et al., J. Bacteriol., 169:4621-4629 (1987); Matoba, et al., Mol. Cell Biol., 8:4904-4916 (1988)). AEP is encoded by the XPR2 gene in Y. lipolytica. Furthermore, large amounts are naturally secreted extracellularly.
Nicaud et al. (Current Genetics, 16:253-260 (1989); EP 0402226 A1) reported chimeric expression of the S. cerevisiae SUC2 with a Y. lipolytica XPR2 promoter and its signal sequence, which resulted in a sucrose-utilizing (SUC+) phenotype in Y. lipolytica. Specifically, 23 N-terminal amino acids from XPR2 were fused to a truncated SUC2 (wherein the truncation removed the first 4 amino acids of the full-length protein). It was reported that about 10% of the invertase activity was observed in the culture broth (i.e., via extracellular secretion), whereas 90% of the activity was recovered using whole cells (i.e., invertase was secreted into the periplasm). Thus, the efficiency in extracellular sucrose hydrolysis was relatively low.
The methodology described by Nicaud et al. has been utilized by others, in their efforts to develop transformant Y. lipolytica strains producing citric acid using sucrose as a carbon source (Wojtatowicz, M., et al., Pol. J. Food Nutr. Sci., 6/47(4): 49-54 (1997); Förster, A. et al., Appl. Microbiol. Biotechnol., 75:1409-1417 (2007); Lazar, Z. et al., Bioresour. Technol., 102:6982-6989 (2011)). Foster et al., above, reported that the majority (60-70%) of invertase activity was found on the cell surface (i.e., cell-bound activity detectable in whole cells, whereas 30-40% of the invertase was detectable in the cell-free culture medium; maximal invertase yield from biomass was 110 U/g dry weight biomass. Most recently, Lazar et al., above, identified a Y. lipolytica strain containing two copies of a fusion comprising the Y. lipolytica XPR2 promoter and its signal sequence and the S. cerevisaie SUC2 and demonstrated that most of the invertase activity was associated with the cells (2568 to 3736 U/g of cells), while about 232 to 589 U/g was extracellular (i.e., only 5-20% of the activity was extracellular).
Thus, engineering Y. lipolytica to have improved extracellular invertase activity is desirable, for it to better utilize sucrose as a carbon source.